Glycosylation of immunoglobulin G (IgG) is recognized as a key modulator of cellular effector functions. At the same time, an increasing body of evidence underlines the importance of other antibody isotypes, especially IgA and IgM, in pathophysiological conditions. Therefore, methods to efficiently study the complex interplay between isotypes, subclasses, and glycosylation of antibodies during acute and chronic states of inflammation are needed. As a solution, we present an integrated and comprehensive method combining simultaneous affinity enrichment of IgG, IgA, and IgM with a single measurement, glycopeptide-centered LC-MS analysis of all isotypes which provides protein-specific (isotype and subclass), and site-specific N- and O-glycosylation quantitation. A two-protease approach provided individual peptides for each glycosylation site, allowing unambiguous compositional assignment and relative quantitation of glycoforms on the MS